Nematicide composition with a synergistic effect, use of nematicide composition with a synergistic effect

ABSTRACT

The present invention relates generically to compositions used for controlling agricultural pests and, in a particular embodiment, to compositions usable for combating nematodes. 
     The invention presents a feasible alternative to the use of nematotoxic compounds that are harmful to the environment and that may damage human health. This alternative is embodied through a nematotoxic composition for controlling nematodes and that may be used on plants, parts of plants and on the soil. The respective technology results from unexpected results achieved from experiments carried out with extracts of the plant  Canavalia ensiformis , resulting in an unheard-of nematotoxic composition comprising a specific combination of substances that are naturally found in the extract and that act synergistically.

FIELD OF THE INVENTION

The present invention falls into the context of green chemistry andrelates generically to compositions used for controlling agriculturalpests and, in a particular embodiment, to compositions usable incombating nematodes.

DESCRIPTION OF THE PRIOR ART

Within the concept of sustainability, environmental chemistry and/orgreen chemistry has advanced toward introducing processes and productsfor replacement of polluting technologies. The use of raw-materials ofvegetable origin and/or substances identical to those found in natureand the incorporation thereof into ecologically correctprocesses/products has proved to be a worldwide tendency, chiefly incountries that have great availability of biomass.

In Brazil, gall-nematodes (Meloidogyne javanica nematodes) figure amongthe most harmful pests in intensive agricultural areas for importantcrops, such as coffee beans, sugar-cane, soybeans, beans, tobacco, fruittrees and olericultural plants. The damages caused by thesephytopathogens include formation of galls that cause unbalance in theabsorption of water and nutrients from the soil, making the plants muchmore sensitive to any environmental modification or to the attack bypests and pathogens. Nematodes of the genus Meloydogyne spp aresedentary endoparasites compulsory on various plants. The hosts compriseover 3,000 vegetable species. Annual losses related to the action ofthese organisms approach US$150 billion, of which 90% are associated tothe Meloigogyne incognica, M. Javanica, M. arenaria and M. haplanematodes (Abad, et al, 2008; Bakhetia. et al, 2005). In this context,M. incognita nematode stands out as the most expressive species as toeconomic losses (Huang et al, 2006).

The strategies used for controlling this phytoparasite consists of:rotation of crops, use of resistant varieties, integrated handling ofpests and chiefly application of synthetic agricultural defensives(Chitwood, 2002). The use of antagonist plants in rotation-of-cropsschemes is one alternative, but with reduction in the productivity andin the profits of the farmer (Dufour et al, 2003; Ferraz & Freitas,2000). The use of resistant varieties is a recommendable way ofcontrolling pests and diseases, but the number of resistant cultivars islimited, as well as the genetic diversity related to this resistance(Dofour et all, 2003); Ferraz & Freitas, 2000). The integrated handlingof pests is a desired strategy, but it needs strict planning, intensivemanagement of the crop, higher expenditure of time and may have costshigher than the use of agricultural defensives (Gentz, 2010). The mostwidely used control strategy lies in the massive use of syntheticnematicides, which result in impairing the natural environment andpublic health (Abad et al, 2008). In this regard, the employ naturalnematotoxic vegetable compounds (Newman et al, 2003) is a strategy forcontrolling this phytonemadode.

Significant advances in the metabolomics area of plants, and theintegration thereof with genomics, transcriptomics, proteomics andbioinformatics have provided new possibilities for exploiting andextending complex interactions ion biological systems ((Weckwerth, 2009;Shulaev, 2008; Weckwerth, 2008; Huang, et al., 2008; Roessner et al.,2006; Roessner et al., 2001). Studies on the action of extracts obtainedfrom seeds of antagonistic plants demonstrated quite effectivenematicidal and nematostatic effects. These effects have been related tothe presence of secondary metabolites, such as alkaloids, terpenes,tannins, flavonoids and glycosides (Chitood, 2002; Ferraz & Freitas,2000).

Thus, it is of extreme importance to look for innovative naturalchemical substances that have wide structural and biological diversity(Newman et al, 2003; Kirkpatrick, 2002), with a view to obtain neweffective and environmental safe nematotoxic products. In this case, theplants are sources of over 100,000 natural products of small molecularmass, known as vegetable compounds or metabolites (Clardy, 2004; Dixon,2001). In vegetables, these compounds may be associated to cellulardifferentiation, regulation of growth, measurement of interactionsbetween plants and other organisms, and chiefly protection of the plant(Dobson, 2004).

Within this context, the present invention presents a feasiblealternative to the use of namatotoxic compounds that are harmful to theenvironment and that may damage human health. This alternative isembodied through a nematotoxic composition for controlling nematodesthat that can be used on plants, parts of plants and in the soil. Therespective technology comes from unexpected results achieved fromexperiments carried out with extracts of Canavalia ensiformis plant,resulting in an unheard of nematotoxic composition comprising a specificcombination of substances that are naturally found in the extract.

SUMMARY OF THE INVENTION

The present invention presents a composition comprising a specificcombination of substances found naturally and that act synergisticallyon the control of nematodes on plants.

The synergistic nematotoxic composition of the present inventioncomprises, as active compounds:

-   -   Trans-aconitic acid,    -   Glucose, and optionally    -   S-carboxymethylcysteine.        Among the forms of presentation of the present composition are        solutions, emulsions, wettable powders, suspensions, powders,        dusts, pastes, soluble powders, granules, suspension-emulsion        concentrated, natural and synthetic materials impregnated with        active compounds and microencapsulations in polymeric        substances. Additionally, the composition of the invention may        further comprise formulation ingredients, including, among        others, liquid carriers/liquid solvents and/or solid carriers        and/or surfactants and/or stickiness agents and/or dyes.

A second embodiment of the respective invention relates to the use ofthe synergistic nematotoxic composition for controlling nematodes onplants.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1—effect of the aqueous crude extracts of seeds of antagonisticplants on (J2) of M. incognita. The bars represent the percentage oflive nematodes after 48 hours' exposure. Concentration of 1 mg of eachextract for a final volume of 0.3 mL and 100 second-stage juveniles wasused in the bioassay in triplicate, having distilled water as negativecontrol.

FIG. 2—Effect of 0.5 mg (0.5 mg/0.3 ml final volume) of the external(DECE) and internal (DICE) dialysates on J2 of M. incognita. The barsrepresent the percentage of dead nematodes. Controls were carried withdistilled water (dH2O), bovine serum albumin (BSA) and aqueous crudeextract of C. ensiformis (ECACe). Recovery assay carried out forcertification of the nematicidal effect.

FIG. 3—Effect of the pre-heated (50° C.) DE of C. ensiformis versus J2of M. incognita, demonstrating the thermal stability of the analyzedfraction. The bioassay was carried out in triplicate, and the barsrepresent the percentage of dead nematodes. Distilled water was used asnegative control. Recovery assay carried out for certification of thenematicidal effect.

FIG. 4—Effect of 500 μg/0.3 mL) of DE of C. ensiformis on saprophyticnematodes of free life (FL/VL) and J2 of M. incocnita. The barsrepresent the percentage of dead nematodes. The controls of the bioassayhave nematodes (VL) and J2 of M. incognita immersed separately indistilled water.

FIG. 5—Test of hemolytic activity using bovine blood (A) or purifiederythrocytes (B) incubated with different concentrations of externaldialysate of C. ensiformis solubilized in PBS. The measurements ofhemolytic activity were monitored spectrophotometrically by using 567-nmwavelengths. Distilled water (100% hemolysis) and PBS were used aspositive and negative controls.

FIG. 6—Effect of the external dialysate (DE) of C. ensiformis on J2 ofM. incognita in greenhouse simulating field conditions. The barssymbolize the number of egg masses found in the roots of tomato-plantsafter the treatments. The experiment was conducted in quintuplicate withdistilled water and the commercial nematicide being used as negative andpositive controls, respectively.

FIG. 7—Optical microscopy: (A) J2 before adding DE-200x. (B) Intestinalregion of J2 before adding DE-400x. (C) J2 24 hours after addingDE-200x. (D) Intestinal region of J2 after adding DE-400x.

FIG. 8 (A) Chromatography of the external dialysate (DE) of C.ensiformis (A=216 nm) exhibiting a profile with 22 peaks. (B)—Bioassayshowing the active fractions against J2 of M. incognita (1, 3, 6, 17 and18) after 48 hours' exposure. (C)—Recovery assay certifying thenematicidal effect of the fractions 1, 3 and 18 and nematostatic effectof fractions 6 and 17.

FIG. 9—(A) Bioassay showing the effect of the commercial compounds onsecond-stage juveniles of M. incognita after 48 hours' exposure.1—Control dH20, 2—DECE, 3—Glycose 10%, 4—Glycose 20%, 5—D-Pinitol (80ug), 6—D-Pinitol (150 ug), 7—L-canavanina (80 ug), 8—L-canavanina (150ug), 9—palmitic acid (80 ug), 10—palmitic acid (150 ug), 11—citric acid(80 ug), 12—citric acid (150 ug), 13—malic acid (80 ug), 14—malic acid(150 ug), 15—cis-aconitic acid (80 ug), 16—cis-anonitic acid (150 ug),17—trans-anonitic acid (80 ug), 18—trans-aconitic acid (150 ug), 19—50mM Tris/HCl pH 8, 20—50 mM acetic acid pH 5, 21—L-phenylamine (80 ug),22—L-phenylamine (150 ug), 23—L-methionine (80 ug), 24—L-methionine (150ug), 25—L-tryptophan (80 ug), 26—L-tryptophan (150 ug),27—S-carboxymethylcistein (80 ug), 28—S-carboxymethylcisteine (150 ug),29—Zantotoxine (80 ug), 30—Zantotoxine (150 ug), and 31—Glucose20%+L-canavanine (15)ug). B—Evaluation of the effect of the differentcommercial compounds after the recovery assay using distilled water.

FIG. 10—(A) Bioassay showing the effect of the commercial compounds andtheir compositions on second-stage juveniles of M. incognita (J1) after48 hours' exposure. C+positive control (extract); C—: negative control(water); 2: palmitic acid; 3: S-carboxymethylcisteine; 4: trans-aconiticacid; 5: glucose. Concentrations of the compositions: 3+4, 3+5=0.25 g/L(each active compound of the composition); 3+4+5=0.17 g/L each activecompound of the composition. (B)—Evaluation of the effect of thedifferent commercial compounds and their compositions after the recoveryassay using distilled water. C+positive control (extract); 2: palmiticacid; 3: s-carboxymethylcisteine; 4: trans-aconitic acid; 5: glucose.Concentrations of the compositions: 3+4, 3+5, 4+5=0.25 g/L (each activecompound of the composition); 3+4+5=0.17 g/L each active compound of thecomposition.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is the result of research works carried out with aview to detect possible natural nematotoxic compounds present in plantextracts. Such compounds constitute an alternative in replacingnematototic synthetic agents that cause damages to nature and to man.The nematotoxic composition described in the invention was generatedunexpected results upon carrying out nematotoxicity bioassays using asactive compounds a few substances detected in the plant extract. Theresults of these bioassays prove the existence of an unexpectedsynergistic nematotoxic effect.

In this context, the present invention describes a nematotoxiccomposition of synergistic effect comprising, as active compounds:

-   -   Trans-aconitic acid,    -   Glucose and optionally    -   S-carboxymethylcisteine.

The nematotoxic composition described in this invention mayalternatively comprise formulation ingredients. Said composition, withor without the presence of formulation ingredients, may be in the formof solutions, emulsions, wettable powders, suspensions, powders, dusts,pastes, soluble powders, granules, suspension-emulsion concentrated,natural and synthetic materials impregnated with active compounds andmciroencapsulations in polymeric substances.

Among the compounds that can be used as formulation ingredients are,among others: liquid carriers/liquid solvents and/or solid carriersand/or surfactants and/or stickiness agents and/or dyes. If theformulation ingredient used is water, it is also possible to employauxiliary solvents such as organic solvents.

Among liquid carriers/liquid solvents suitable for use in the presentinvention, one can cite, among others, aromatic solvents, chlorinatedaromatic solvents or chlorinated aliphatic hydrocarbons, aliphatichydrocarbons, petroleum fractions, mineral oils, vegetable oils,alcohols and their ethers and esters, ketones, strongly polar solventsand water.

Preferably, the aromatic solvents used in the invention are selectedfrom xylene, toluene or alkyl naphtalenes. In the case of the use ofchlorinated aromatic solvents or chlorinated aliphatic hydrocarbons inthe composition of the present invention, these are preferably selectedfrom chlorobenzene, chloroethylene or dichloromethane. Aliphatichydrocarbons are preferably selected from cyclohexane or paraffins. Inthe alternative case of using alcohols, these are preferably selectedfrom ethanol, propanol butanol or glycol. For the case of using ketones,these are preferably selected from propanone, methyl ethyl ketone,methyl isobutyl ketone or cyclohexanone. Strongly polar solvents arepreferably selected from dimethylformamide and dimethyl sulfoxide.

Among the solid carriers suitable for use in the present invention, onecan cite, among others, ammonium salts, ground natural minerals andground synthetic minerals.

Preferably, when ground natural minerals are used as solid carriers,they are selected from kaolin, clays, talc, chalk, quartz, atapulgite,montmorilonite and diatomaceous earths. When using ground syntheticminerals, these are preferably selected from silica, alumina andsilicates. Solid carriers suitable are used when the nematotoxiccomposition of the present invention is presented in the form ofgranules. These suitable solid carriers are preferably selected fromground natural rocks, synthetic granules and organic material granules.

Alternatively, surfactants may be employed as formulation ingredientsfor the composition of the present invention. The respective surfactantsare selected from emulsifiers and/or dispersants and/or foam builders.Different emulsifiers/foam builders may be used in the nematotoxiccomposition of the present invention, including cationic, non-ionic andanionic emulsifiers. When using non-ionic and anionic emulsifiers, theseare preferably selected from fatty polyoxyethylene esters, fattypolyoxyethylene ethers, alkyl sulfonates, alkyl sulfates, arylsulfonates, and protein hydrolysates. In the case of using dispersantssuch as surfactants in the present invention, these are preferablyselected from methylcellulose and lignin sulfite waste.

Other formulation ingredients may also, alternatively, be employed inthe composition of the present invention. These ingredients include,among others, stickiness agents such as carboxymethylcellulose andnatural and synthetic polymers in the form of powders, granules orlatex, such as gum Arabic, polyvinyl alcohol and polyvinyl acetate.Other ingredients that may also be employed as formulation ingredientsare mineral and vegetable oils, natural phospholipids such as cephalinslecithins, and synthetic phospholipids.

Alternatively, the nematotoxic composition of the present invention mayalso contain, as formulation ingredients, dyes such as inorganicpigments, such as iron oxide, titanium oxide and Prussia blue, andorganic dyes such as alizarin dyes, azo dyes and metallic phthalocyaninedyes.

In an alternative embodiment of the present invention, the nematotoxiccomposition of synergistic effect may further comprise other activecompounds. These other active compounds, when used, are selected frominsecticides, attractors, sterilizing agents, bactericides, acaricides,fungicides, growth regulating substances, herbicides and safeners.

In another alternative embodiment of the present invention, thesynergistic nematotoxic composition is an aqueous composition comprisingthe active compounds. In a preferred particular composition of thepresent invention, the nematotoxic composition is an aqueous compositioncontaining palmitic acid and trans-aconitic acid in concentration of0.25 g/L each. In a second preferred particular composition of thepresent invention, the nematotoxic composition is an aqueous compositioncontaining palmitic acid and glucose in concentration of 0.25 g/L each.In a third preferred particular composition of the present invention,the nematotoxic composition is an aqueous composition containingpalmitic acid, tans-aconitic acid and glucose ion concentration of 0.17g/L. In a fourth preferred particular composition of the presentinvention, the nematotoxic composition is an aqueous compositioncontaining palmitic acid, trans-aconic acid and S-carboxymethylcisteinein concentration of 0.17 g/L each. In a fifth preferred particularcomposition of the present invention, the nematotoxic composition is anaqueous composition containing palmitic acid, glucose andS-carboxymethylcisteine ion concentration of 0.17 g/L each. In a sixthpreferred embodiment of the present invention, the nematotoxiccomposition is an aqueous composition containing palmitic acid, glucose,S-carboxymethylcistein and trans-aconitic acid in concentration of 0.125g/L each.

Another object of the present invention is the use of the nematotoxiccomposition of synergistic effect described before for controllingnematodes on plants. Particularly, said use takes place by applying thecomposition of the invention to the plant and/or parts thereof and/or tothe soil.

The application of the synergistic nematotoxic composition of thepresent invention to the plant and/or parts thereof and/or the soil maybe carried out either directly or by allowing the compounds to act ontheir vicinities, environment or storage space by the ordinary treatmentmethods, for example, by immersion, spraying, evaporation, mist,spreading, etc.

In a special embodiment of the present invention, the use of thenematotoxic composition of synergistic effect described in this documentis for controlling Meloigonyne incognita.

Experiments Carried Out and Results Achieved:

Initially, aqueous extracts obtained from seeds of antagonistic plantswere tested in bioassays with a view to detect compounds effectiveagainst second-stage juveniles (J2) of M. incognita.

Among the extracts tested, Canavalia ensiformis, a plant of the familyLeguminosae, originating in Central America, exhibited the highestnematicidal activity (85%) (FIG. 1). The fractioning of this material bydialysis resulted in the separation of molecules smaller than 3.5 kDa(external dialysate—DE) and bigger than 3.5 kDa (internal dialysate—DI).In this case, the DE exhibited the most promising results as compared tothe DI, such as: a) higher nematotoxic activity (FIG. 20); b) thermalstability (FIG. 3); c) high specificity against J2 of M. incognita(Table 1); d) absence of activity against fungi (phitopathogenicCollectotrichum gloeosporioides access 1915, Fusarium solani access1624, Macrophomina phaseolina access 1496 and Phytophthora tabacum),bacterium (Bacillus subtilis) and insect larva (Antonomus grandis andSpodoptera frugiperda) (Table 1); e) non-existence of activity againstfree-life nematode (FIG. 4); absence of adverse cytotoxic effect on redcells of mammals (FIG. 5) and g) reduction higher than 80% of the M.incognita egg masses in bioassays conducted in greenhouse by usingtomato plants (FIG. 6).

Additionally, the viewing by optical microscopy of the effect caused bythe compound (s) present in the De of C. ensiformis on J2 of M.incognita disclosed the break of the normal anatomy (FIG. 7—A, B, C andD) with formation of various vacuoles along the stiff body of thenematode. The alterations suggest the destructuring of tissues of theintestines of the phytoparasite.

TABLE I Results achieved in the assays for specificity conducted invitro by using different concentrations of DE of C. ensiformis againstfungus, bacterium, insect larvae and nematode. The bioassays we3ecarried out ion triplicate. A = Fungus, B = Bacterium, C = Larvae ofinsect and D = Nematode μg of DE of C. ensiformis/ Pathogen BioeassayActivity A Colletotriclum 250,500,1000 Innocuous - completegloeosporinoides absence of halo A Fusarium solani 250,500,1000Innocuous- complete absence of halo A Macrophomina 250,500,1000Innocuous - complete phaseoline absence of halo B Bacillus subtilis250,500,1000 Innocuous - complete absence of halo A Anthonomus grandis1000 Innocuous - complete (Larva) absence of halo C Soidioterafrugiperda 1000 Innocuous - complete (Larva) absence of halo D Dematoidesaprofitico 500 Innocuous D Dematoidogine 500 Nematicidal incognita

The purification of the DE via HPLC using reverse column phase (C18enabled the separation of three nematicidal fractions and twonematostatic ones. All the steps were monitored by bioeassays forevaluation of the nematotoxic activity (FIG. 8 A, B and C). The activefractions were analyzed by Metabolite profiling by using the techniquesof gaseous chromatography coupled to mass spectrometry (GC-MS), liquidchromatogeraphy coupled to mass spectrometry (LC-MS) and nuclearmagnetic resonance (1H RMN). The data generated by these technologiesadded to a vast research in the literature enabled identification ofnine compounds with nematotoxic action.

In this context, compounds produced commercially with biologicalfunctions identified after research in the literature and present in thenematotoxic fractions of C. ensiformis were tested in bioassay againstM. incognita juveniles (FIG. 9A). Glucose solution containing 10% (W/V)was capable of paralyzing most of the nematodes after a period ofexposure of 48 hours (FIG. 9A). After the recovery assay, 87% of the J2reestablished the mobility, confirming the nematostatic activity for theconcentration of applied glucose (FIG. 9A). However, glucose solutionscontaining 20% (W/V) exhibited a nematicidal effect killing over 90% ofthe J2 after 48 hours exposure (FIG. 9B). The vegetable carbohydrateD-pinitol did not exhibit any toxic effect after 48 hours' exposureagainst J2 of M. incognita, using concentrations of 80 μg/300 μL and 150μg/300 μL, respectively (FIG. 9A). Similarly, the non-protein amino acidL-canavanine did not exhibit any nematotoxic activity for concentrationof 80 μg/300 μL after 48 hours' exposure (FIG. 9A). However, when theconcentration is increased to 105 μg/300 μL, 25% of the juvenilesremained paralyzed after the recovery assay, thus confirming a modestnematicidal activity (FIG. 9B). Among the organic acids: malic, citric,cis-aconitic, trans-aconitic and palmitic, only the latter two acidswere capable of paralyzing 90 and 98% of the J2 of M. incognita forconcentration of 80 μg/300 μL after 48 hours' exposure (FIG. 9A). Theother organic acids paralyzed a very low percentage of J2 for the sameconcentration and exposure time (FIG. 9A). For concentration of 150μg/300 μL, the transaconitic acid exhibited nematicidal activity of 98%,while the malic, citric and cis-aconitic acids exhibited a nematicidaleffect of only 18, 25 and 40% after the recovery assay (FIG. 9B). On theother hand, the palmitic acid demonstrated a nematostatic effect forboth concentrations evaluated with 90% of the J2 recovering the mobilityafter the recovery test (FIG. 9B). These results show clearly adose-dependent effect of the organic acids tested. The amino acidsL-phenylamnine and L-triptofane were not capable of affecting the J2using concentrations of 80 μg/300 μL and 150 μg/300 μL, respectively(FIG. 9A). Inversely, the compound S-carboxymethylcisteine paralyzedabout 90% of the juveniles after 48 hours' exposure for bothconcentrations of 80 μg/300 μL and 150 μg/300 μL (FIG. 9A). Recoveryassay confirmed the nematostatic effect with most J2 recovering themovement (FIG. 89A). Xantotoxin paralyzed 87% of the J2 after 48 hours'exposure for both the concentration of 80 μg/300 μL and theconcentration 150 μg/300 μL (FIG. 9A). The J2 subjected to the recoveryassay using both concentrations did not recover the mobility, confirmingthe nematicidal effect (FIG. 9B).

With a view to obtain a nematotoxic composition from the compounds withnematotoxic activity, detected in the prior experiments, the compoundsglucose (5), trans-aconitic acid (4) and S-carboxymethylcisteine (3)were tested by carrying out in vitro experiments in differentcombinations. Although of xantotoxine demonstrates nematicidal action,it was not selected for aid tests due to the high cost and thedifficulty of its chemical synthesis.

A few compositions containing combinations of active compounds weretested by carrying out in vitro bioassays against M. incognitajuveniles. The active compounds used in the experiments carried out wereobtained commercially.

The compositions tested were:

-   -   a) Trans-aconitic acid+glucose (0.25 g/L of each compound);    -   b) Glucose+S-carboxymethylcisteine (0.25 g/L of each compound);    -   c) S-carboxymethylcisteione+trans-aconitic acid (0.25 g/L of        each compound;    -   d) Trans-aconitic acid+glucose+S-carboxymethylcisteine (0.17 g/L        of each compound).        The results achieved through the tests carried out with the        compositions a), b), and d) are shown in FIGS. 10 (A) and (B)        and commented on hereinafter.

As can be seen in FIG. 10A, the compositions a) and d) unexpectedlyexhibit a synergistic nematotoxic effect as with regard to theparalyzation of the nematodes, as compared to the sum of the resultsobtained individually by each active compound. Said comparisons areshown in Tables 2 and 3 below.

TABLE 2 Synergistic nematotoxic effect obtained after tests with thecomposition a) Active compound or Concentration % of paralyzedcomposition (g/L) nematodes Trans-aconitic acid 0.25 27.08 Glucose 0.250.71 Glucose + trans- 0.25 (of each 61.2 aconitic acid compound)compositionThe analysis of the results obtained in the recovery assays and shown inFIG. 10B shows that the synergistic nematotoxic effect obtained bycomposition a) corresponded to a nematostatic effect.

TABLE 3 Synergistic nematotoxic effect obtained after tests withcomposition d) Active compound of % paralyzed composition Concentration(g/L) namatodes Glucose 0.17 0 S- 0.17 43.03 carboxymethylcisteineTrans-aconitic acid 0.17 11.12 Glucose + S- 0.17 (of each 97.39carboxymethylcisteine + compound) trans-aconitic acid compositionThe analysis of the results obtained in the recovery assays and shown inFIG. 10B shows that the synergistic nematotoxic effect obtained bycomposition b) corresponded to a nematostatic effect.

The nematotoxic compositions b) and c), the in vitro assay results ofwhich are also shown in FIGS. 10A and 10B and do not exhibit asignificant synergistic nematotoxic effect.

1. A nematotoxic composition of synergistic effect, characterized bycomprising, as active compounds: Trans-aconitic acid; Glucose, andoptionally S-carboxymethylcisteine.
 2. The nematotoxic composition ofsynergistic effect according to claim 1, characterized by comprisingformulation ingredients.
 3. The nematotoxic composition of synergisticeffect according to claim 1, characterized by being in the form ofsolutions, emulsions, wettable powders, suspensions, powders, dusts,pastes, soluble powders, granules, suspension-emulsion concentrates,natural and synthetic materials impregnated with the active compoundsand microencapsulations in polymeric substances.
 4. The nematotoxiccomposition of synergistic effect according to claim 1, characterized inthat the formulation ingredients are liquid carriers/liquid solventsand/or solid carriers and/or surfactants and/or stickiness agent and/ordyes.
 5. The nematotoxic composition of synergistic effect according toclaim 4, characterized ion that the liquid carriers/liquid solvents areselected from aromatic solvents, chlorinated aromatic solvents orchlorinated aliphatic hydrocarbons, aliphatic hydrocarbons, petroleumfractions, mineral oils, vegetable oils, alcohols and their ethers andesters, ketones, strongly polar solvents and water.
 6. The nematotoxiccomposition of synergistic effect according to claim 5, characterized inthat the aromatic solvents are selected from xylene, toluene oralkylnaphthalenes.
 7. The nematotoxic composition of synergistic effectaccording to claim 5, characterized in that the chlorinated aromaticsolvents or chlorinated aliphatic hydrocarbons are selected fromchlorobensene, chloroethylene or dichloromethane.
 8. The nematotoxiccomposition of synergistic effect according to claim 5, characterized inthat the aliphatic hydrocarbons are selected from cyclohexane orparaffins.
 9. The nematotoxic composition of synergistic effectaccording to claim 5, characterized in that the alcohols are selectedfrom ethanol, propanol, butanol or glycol.
 10. The nematotoxiccomposition of synergistic effect according to claim 5, characterized inthat the ketones are selected from propanone, methyl ethyl ketone,methyl isobutyl ketone or cyclohexanone.
 11. The nematotoxic compositionof synergistic effect according to claim 5, characterized ion that thestrongly polar solvents are selected from dimethylformamide and dimethylsulfoxide.
 12. The nematotoxic composition of synergistic effectaccording to claim 4, characterized in that the solid carriers areselected from ammonium salts, ground natural minerals and groundsynthetic minerals.
 13. The nematotoxic composition of synergisticeffect according to claim 12, characterized in that the ground naturalminerals are selected from kaolin, clays, talc, chalk, quartz,atapulgite, montmorilonite and diatomaceous earth.
 14. The nematotoxiccomposition of synergistic effect according to claim 12, characterizedin that the ground synthetic minerals are selected from silica, aluminaand silicates.
 15. The nematotoxic composition of synergistic effectaccording to claim 4, characterized in that the solid carriers forgranules are selected from ground natural rocks, synthetic granules andorganic material granules.
 16. The nematotoxic composition ofsynergistic effect according to claim 4, characterized in that thesurfactants are emulsifiers and/or dispersants and/or foam builders. 17.The nematotoxic composition of synergistic effect according to claim 16,characterized in that the emulsifiers are selected from cationic,non-ionic and anionic emulsifiers.
 18. The nematotoxic composition ofsynergistic effect according to claim 17, characterized in that thenon-ionic and anionic emulsifiers are selected from polyoxyethyele fattyesters, polyoxyethylene fatty ethers, alkyl sulfonates, alkyl sulfates,aryl sulfonates, and protein hydrolysates.
 19. The nematotoxiccomposition of synergistic effect according to claim 16, characterizedin that the dispersants are selected from methylcellulose and ligninsulfite waste.
 20. The nematotoxic composition of synergistic effectaccording to claim 4, characterized in that the stickiness agents areselected from carboxymethylcellulose, natural polymers and syntheticpolymers.
 21. The nematotoxic composition of synergistic effectaccording to claim 4, characterized in that the dyes are selected frominorganic pigments and organic dyes.
 22. The nematotoxic composition ofsynergistic effect according to claim 1, characterized by comprisingother active compounds.
 23. The nematotoxic composition of synergisticeffect according to claim 22, characterized ion that the other activecompounds are selected from insecticides, attractors, sterilizingagents, bactericides, acaricides, namaticides, fungicides,growth-regulating substances, herbicides and safeners.
 24. Thenematotoxic composition of synergistic effect according to claim 1,characterized in that the composition is an aqueous composition.
 25. Thenematotoxic composition of synergistic effect according to claim 24,characterized in that the aqueous composition contains trans-aconiticacid and glucose in concentration of 0.25 g/L each.
 26. The nematotoxiccomposition of synergistic effect according to claim 24, characterizedion that the aqueous composition contains S-carboxymethylcisteine,trans-aconitic acid and glucose in concentration of 017 g/L each. 27.Use of the nematotoxic composition of synergistic effect defined inclaim 1, characterized in that it is for controlling nematodes onplants.
 28. The use of the nematotoxic composition of synergistic effectaccording to claim 27, characterized in that it is applied to the plantand/or to parts of the plant and/or to the soil.
 29. The use of thenematotoxic composition of synergistic effect according to claim 27,characterized in that it is for controlling Meloigogyne incognita.